LNCaP cells are maintained in androgen-deprived conditions for 24 hours and treated with 10 μM Compound 30 or MDV3100 for 2 hours followed by addition of 1 nM of R1881. After 15 minutes incubation, cells are fixed and AR localization is assessed by immunofluorescence imaging.

CLU is induced by MDV3100 in time- and dose-dependent manner. LNCaP cells are treated with MDV3100 at indicated time (left) or concentration (right) and Western blot analysis is conducted with CLU, AR, and PSA antibodies.

MDV3100-induced CLU is mediated via p90Rsk-YB-1 signaling pathway. MDV3100 activates AKT and MAPK pathways. LNCaP cells are treated with 10 μM MDV3100 for indicated times and Western blotted using CLU, p-Rsk/R/Rsk, p-S6K/S6K, and pYB-1/YB-1. Vinculin is used as a loading control.

LNCaP and DuCaP cells are 1 treated for 72 hours with 1 or 10 μM Enzalutamide in the presence of increasing IL6 concentrations. The effect on JAK/STAT3 signaling is measured by determining STAT3 Tyr705-phosphorylation via Western blot and calculation of dose response curves.

Combination of Enzalutamide and 17-AAG lead to decreased AR protein level and transcriptional activity. (A&B) LNCaP (A) and C4-2 (B) cells are treated as indicated for 24 hr, followed by IB against AR, PSA and CHIP. (C&D) 22RV1 (C) and MR49F (D) cells are treated as indicated for 24 hr, followed by IB against AR and HSP90. (E) C4-2 cells are treated as indicated for 24 hr, fractionated into cytoplasm and nuclear, followed by IB against AR and Plk1.

Effects of culture in CSS and treatment with Enzalutamide on cell cycle of T24GR cells. T24 and T24GR cells (5x105) are seeded in a 6-well plate and cultured for 24 h. The culture medium is changed to that containing 50 μM Enzalutamide or vehicle (DMSO). Seventy-two hours later, cell lysates are harvested and subjected to western blot analysis for cyclin B1, D1 and β-actin.

Short term treatment (14 days) of LAPC4 vehicle cells with 8μM Enzalutamide clearly demonstrates that AR overexpression is not a short term effect of drug treatment but develops as a long term adaptation during acquisition of resistance. It has been suggested that presence of a truncated AR variant (AR-V7) is associated with resistance to Enzalutamide.

VCaP (mock) and VCaP (UGT2B17) cells are cultured in the RPMI1640 medium plus 5% CSS. Cells are treated with vehicle, 10 nM of R1881 or 10 μM of Enzalutamide (ENZ) for 0 or 28 days. Protein levels of UGT2B17, pSrc, tSrc, pAKT, total AKT, pSTAT3, total STAT3, pSTAT5, total STAT5 and β-actin are determined by immunoblotting.

LNCaP cells are cultured in RPMI1640 medium containing 5% CSS and treated with vehicle, 1 μM of ICRF187 or 1 μM of ICRF193 in addition to vehicle, 10 nM of R1881 or 10 μM of ENZ treatment for 2 hours. Three independent ChIP experiments are performed using the AR antibody. AR protein levels under 2 and 24 hour treatment are detected by Western blotting.

Representative images of 22Rv1 cells transduced with PSEBC-TSTA and showing that single cell luminescence increases with exposure time but not the number of detected cells. Bioluminescence microscopy can titrate AR agonist DHT (0.5-10 nM) concentration ability to activate AR-transcription. LAPC4-GFP cells are infected with PSEBC-TSTA in media containing 0.5 to 10 nM of Dihydrotestosterone (DHT).

p-TNIK (S764), T-TNIK, and PSA protein expression are assessed in (A) LNCaP and 16DCRPC cells that are treated with 10 μM Enzalutamide (ENZ) in media containing 10% FBS for 2, 4, and 7 days by western blot. Vinculin is used as loading control.

Enzalutamide is dissolved in DMSO and stored, and then diluted with appropriate medium before use[1].

LNCaP cells (107 cells/condition) are grown in RPMI media supplemented with 5% charcoalstripped serum for 22 days, then treated with DMSO or 1 nM R1881, combined with an antiandrogen (DMSO, 1 μM Bicalutamide, 10 μM Bicalutamide, 1 μM RD162, 10 μM RD162, 1 μM MDV3100, or 10 μM MDV3100) for 8 hours. An aliquot of cells are harvested for qRT-PCR of PSA and TMPRSS2 mRNA. The remaining cells are cross-linked using 1% paraformaldehyde for 10 minutes, then glycine is added and samples centrifuged (4°C, 4000 rpm, 5 minutes) to stop further crosslinking. Chromatin immunoprecipitation is performed using a chromatin immunoprecipitation assay kit. Immunoprecipitated DNA is amplified by real-time PCR. Primers are PSA enhancer forward-ATGTTCACATTAGTACACCTTGCC and reverse-TCTCAGATCCAGGCTTGCTTACTGTC and TMPRSS2 enhancer forward-TGGTCCTGGATGATAAAAAAAGTTT and reverse-GACATACGCCCCACAACAGA[1]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice[3]Following a 5-day acclimation period, 5- to 9-week-old male CB17SCID mice are castrated and allowed to recover for an additional 5 days before inoculation with tumor cells. LNCaP cells co-expressing exogenous AR and the AR-dependent reporter construct ARR2-Pb-Luc (LNCaP-AR-Lux cells) are used to generate a xenograft model of human prostate cancer. Before implantation, LNCaP-AR-Lux cells are prepared by the addition of trypsin-EDTA, washed with complete medium, collected and resuspended at 20×106 cells/mL. Cell suspensions are diluted with Matrigel to 2×106 cells/0.2 mL and delivered subcutaneously in the suprascapular region. Tumor growth is monitored to the volume of 100 mm3 when treatment begins (80 days). The observed rate of tumor take with LNCaP-AR-Lux cells is between 70% and 80%. Body weight and tumor volumes (width2×length/2) are measured two to three times per week with a digital caliper, and the average tumor volumes are determined. Test drugs are diluted in Tween 80:PEG 400, and stored at 4°C until administration by oral gavage. Each group of mice (n=7) is treated daily for 28 consecutive days with 1, 10, or 50 mg/kg Enzalutamide, vehicle control, or 50 mg/kg Bicalutamide. At the end of the treatment period or when tumor volume exceeded 1,000 mm3, animals are euthanized and blood and tissue samples are collected for analysis. Rats[4]Male SD rats (n=3) are administered Enzalutamide through the tail vein (intravenous) and by oral gavage at 1 mg/kg and are kept in metabolic cages after dosing. Urine and feces samples are collected over the following time intervals after dosing: 0-2, 2-4, 4-6, 6-10, 10-24, 24-48, and 48-72 h. The metabolic cages are rinsed with distilled water, and residues are added to the urine samples at 72 h. To extract the Enzalutamide present in the feces, samples are shaken vigorously for 12 h with 50 % methanol. MCE has not independently confirmed the accuracy of these methods. They are for reference only.